David updates meeting
dissertation
- should I be concerned that I haven't heard anything from most of my committee about my draft yet?
- I only heard back from Roberto, who said he couldn't read the draft in detail but suggested some references mostly about CME scission module proteins that don't seem very relevant to my thesis
Mechanical role of Type I myosin in CME theory project
- we learned one last unknown parameter of myosin is pretty important for motor
function: "stiffness" (spring constant of myosin-actin bond). I used simulated
gliding assays to constrain this parameter
- very low stiffness: bonds stretch a lot, not much directed motility
- medium stiffness: some bond stretching, peak motility
- high stiffness (what I had previously guessed in CME simulations): no motility because even myo5-calibrated weak catch bond is engaged and off-rate gets extremely low
Ross's Myo5 gliding motility assay velocity measurements:
- quantification of gliding assay simulation displacements shows that the
medium stiffness most closely recapitulates measured velocity
with newly fit stiffness value, re-ran CME simulations over range of myosin unbinding rate, catch bond activity, and resistance to internalization (this is currently in my dissertation)
- weak catch bond seems very helpful at low resistance, but I think data is incomplete at high resistance (does it become more assistive at stronger catch bond activity, but it's just off the plotted area?)
- currently running expanded set of simulations relative to the last set of
results to sweep the following parameter ranges in 12 replicates
- myosin copy number: 0, 50, 100, 200
- resistance to internalization: 2, 20, 200, 2000
- unbinding rate: 0.01, 0.1, 1, 10, 100, 1000, 10000, 100000
- unbinding force: -0.01, -0.1, -1, -10, -100, -1000, 0
- started simulations several weeks ago while writing my dissertation, but only
recently found out I exhausted our lab's allocation of computing time on the
cluster :(
- following up with Matt to figure out how he was able to get more in the past
- the very high volume of simulation data requires reworking my analysis pipeline to avoid freezing our lab workstation, so I have been busy with that in the meantime
- also currently working out analysis methods to extract the readouts we need
for putting together the story
actin density, filament count, capped vs. uncapped end count
comparison of internalization in different myosin property combinations to the total actin density
- analyis of per-filament elongation rate TBD
- need to figure out if/when my access to computing resources will be restricted after I graduate
Nikhil's protein purification project
- Nikhil is very close to replicating the Stachowiak lab's EPS15 purification
protocol, currently working on optimizing yield and further reducing
degradation
- the smaller size of our gel filtration column might be yield-limiting
example gel filtration fractions from Liping
example gel filtration fractions from Nikhil
- stain-free SDS-PAGE imaging system works on the 5th floor imager, so we can speed up the chromatography fraction analysis steps
- I trained Leanna and Nikhil on making bead SLBs, she figured out how to adapt
it to coverslip SLBs and trained us on that
- this is necessary for passivating coverslips for in vitro droplet experiments
- trained Nikhil on airyscan FRAP; confirmed that SLBs he made on beads and coverslips are fluid
- Nikhil currently troubleshooting NHS ester fluorescent dye labeling on EPS15
- the spin column that the Stachowiak lab used to use for removing unbound dye is discontinued and they haven't figured out an alternative yet
- Nikhil is experimenting with dialysis to balance labeling efficiency with protein degradation
- hoping to try first droplet experiment with purified EPS15 together next week before I leave
- handing off direct mentorship of Nikhil to Leanna, but I will stay involved in ~monthly meetings to guide his project on a higher level